Genetic, quantitative and microscopic evidence for fusion of haploid nuclei and growth of somatic calli in cultured ms10 35 tomato anthers

Abstract

In plant breeding, androgenic doubled haploids represent powerful tools to save time and resources for pure line generation. While in many species efficient protocols are known, in tomato (Solanum lycopersicum), the knowledge on the induction of androgenesis is still very scarce, and little is known about the particularities of this highly recalcitrant species. The only known method capable of yielding haploid/doubled haploid tomato plants is anther culture. However, this method has important limitations, including low efficiency of haploid induction and a low proportion of spontaneously doubled haploids. To understand these limitations better, we have analyzed the process of callus formation in anthers of tomato lines carrying the ms10 35 gene for male-sterility, using light and electron microscopy, flow cytometry and genetic analysis with morphological and molecular markers. Our results demonstrate that haploid, doubled haploid and diploid calli occur in tomato anthers, although at different frequencies. Diploid calli derived either from somatic cells or from the fusion of two genetically different haploid nuclei account for more than 90% of the total of calli produced. Somatic calli are derived from the stubs of connective tissue present in the interlocular septa of anthers. This growth is markedly increased in the ms10 35 mutants, which explains their higher callogenic rates than standard tomato lines. Together, our results reveal serious drawbacks that explain the low efficiency of anther-derived, doubled haploid production in tomato, and stress the need for alternatives towards doubled haploidy.

Assessment of different anther culture approaches to produce doubled haploids in cucumber (Cucumis sativus L.)

“…In light of our results and previous reports, it seems evident that cucumber is a strongly recalcitrant species where conventional protocols involving mild stresses and few or no growth regulators in the culture medium are unlikely to work. As also shown for other heavily recalcitrant species such as tomato (Seguí-Simarro and Nuez 2007;Corral-Martínez et al 2011), a combination of different stresses together with growth regulators appear necessary to promote the initial microspore cell proliferation. Thus, we propose that at least for the particular cases of Esfahani and Beta Alpha, the most efficient protocol to produce DHs at present involves a first step of callus induction through bud pretreatment at 4ºC and the application of a 35ºC heat shock to anthers, once inoculated in medium with 0.68-0.91 mg/l BAP and 1 mg/l 2,4-D. With this, a maximum yield of 6.67% (6.67 diploid/DH calli/100 anthers) was achieved.…”

“…We used flow cytometry to assess the ploidy level of donor plant leaves (as standards for 2C DNA content), induced calli and regenerated plants as described in Corral-Martínez et al (2011). Small pieces of cultured calli and leaves were processed using the CyStain UV Precise P kit (Partec GmbH, Münster, Germany).…”

“…The inner side is the side of filament insertion. It is known that the wound left after filament excision is especially prone to proliferate, producing high rates of somatic calli in anther cultures of different species (Seguí-Simarro and Nuez 2006;Corral-Martínez et al 2011). Thus, it is quite possible that filament remnants are also exerting a strong influence in this case.…”

The origin of clones among androgenic regenerants of hexaploid triticale

“…It is known that in androgenesis of triticale, spontaneous doubling of the chromosome number is relatively common. However, diploids may also originate from somatic tissues of the anther (Corral-Martínez et al 2011;Smykalova et al 2012). Genotyping performed here clearly shows that all regenerants were derived from microspores and were homozygous for specific alleles (the source plants were F 1 hybrids, heterozygous at many tested loci).…”

“…Here, SSR analysis was used for its simplicity, low cost and ability to identify multiple alleles at given loci. It has been used for large scale analyses to identify homozygous regenerants in maize (Belicuas et al 2007), potato (Chani et al 2000), cucumber (Diao et al 2009) and tomato (Corral-Martínez et al 2011).…”